Allele Quantification Pyrosequencing® at Designated SNP Sites to Detect Allelic Expression Imbalance and Loss-of-Heterozygosity.

Pyrosequencing(®) is able to quantitate the level of a nucleotide at a designated germ-line or somatic variant, including single nucleotide polymorphisms (SNPs). SNPs within a gene of interest may be used to distinguish between the two genetic alleles and study their behavior in heterozygous individuals. With regard to cancer etiology and development, identification of alleles and the detection of allelic imbalances, such as transcriptional loss from one allele or loss-of-heterozygosity (due to deletion of one allele), within a tumor are particularly useful. Lynch syndrome, the most common form of hereditary bowel and uterine cancer, is caused by heterozygous germ-line mutations within the DNA mismatch repair genes and tumors develop following inactivation of the remaining functional allele within somatic tissues, usually by acquired loss-of-heterozygosity. MLH1 is the most frequently mutated gene in Lynch syndrome; however, some cases whose tumors display immunohistochemical loss of the MLH1 protein have no apparent mutation within the coding region of MLH1. Allelic loss of expression or reduced function of MLH1 can also result in the propensity to develop Lynch syndrome associated cancers. In this chapter we describe allele quantification Pyrosequencing assays designed at a common benign SNP within the MLH1 coding region for application to either DNA or mRNA (cDNA) templates, which enabled us to detect pathological allelic imbalances in such cases with suspected Lynch syndrome. Our allele quantification Pyrosequencing assays at the MLH1 c.655A > G (rs1799977) exonic SNP were applied to clinical specimens and detected both constitutional allelic expression loss and tumor loss-of-heterozygosity in some cases, facilitating the identification of the mechanistic cause underlying their cancer development. We provide detailed protocols for implementing these Pyrosequencing assays and illustrative examples of their application in patients.